1. Field of the Invention
This invention relates to isocoumarin derivatives and their use in cancer therapy. More particularly, it relates to the use of isocoumarin derivatives in the prevention or treatment of cancer by inhibiting tumor neovascularization, or angiogenesis, in combination with enhancing tumor sensitivity to radiation therapy and/or chemotherapy.
2. Description of Related Art
Normal tissue homeostasis is achieved by an intricate balance between the rate of cell proliferation and cell death. Disruption of this balance either by increasing the rate of cell proliferation or decreasing the rate of cell death can result in the abnormal growth of cells and is thought to be a major event in the development of cancer. The effects of cancer are catastrophic, causing over half a million deaths per year in the United States alone. Conventional strategies for the treatment of cancer include chemotherapy, radiotherapy, surgery, biological therapy or combinations thereof. However, further advances in these strategies are limited by lack of specificity and excessive toxicity to normal tissues. In addition, certain cancers are refractory to treatments such as chemotherapy, and some of these strategies, such as surgery, are not always viable alternatives.
Once the diagnosis of cancer is established, the most urgent question is whether the disease is localized or has spread to lymph nodes and distant organs. The most fearsome aspect of cancer is metastasis, and this fear is well justified. In nearly 50% of patients, surgical excision of primary neoplasms is ineffective, because metastasis has occurred by the time the tumor is large enough for resection (Sugarbaker, 1979; Fidler and Balch, 1987). Metastases can be located in different organs and in different regions of the same organ, making complete eradication by surgery, radiation, drugs or biotherapy difficult. Furthermore, the organ microenvironment significantly influences the response of tumor cells to therapy (Fidler, 1985), as well as the efficiency of anticancer drugs, which must be delivered to tumor foci in amounts sufficient to destroy cells without causing undesirable side effects (Fidler and Poste, 1985). In addition, the treatment of metastatic cancer is greatly hindered due to the biological heterogeneity of cancer cells, and the rapid emergence of tumor cells that become resistant to most conventional anticancer agents (Fidler and Poste, 1985).
One of the processes involved in the growth of both primary and secondary (metastatic) tumors is neovascularization, or creation of new blood vessels which grow into the tumor. This neovascularization is termed angiogenesis (Folkman, 1986; Folkman, 1989), which provides the growing tumor with a blood supply and essential nutrients. Although tumors of 1-2 mm in diameter can receive all nutrients by diffusion, further growth depends on the development of an adequate blood supply through angiogenesis. Inhibition of angiogenesis provides a novel and more general approach for treating metastases by manipulation of the host environment.
Several angiogenic molecules released by both tumor endothelial cells and the normal cells surrounding the tumor endothelial cells mediate the induction of angiogenesis. The prevascular stage of a tumor is associated with local benign tumors, whereas the vascular stage is associated with tumors capable of metastasizing. Moreover, studies using light microscopy and immunohistochemistry concluded that the number and density of microvessels in different human cancers directly correlate with their potential to invade and produce metastasis (Weidner, et al., 1991; Weidner, et al., 1993). Not all angiogenic tumors produce metastases, but the inhibition of angiogenesis prevents the growth of tumor endothelial cells at both the primary and secondary sites and thus can prevent the emergence of metastases.
Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a xe2x80x9csproutxe2x80x9d off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
Persistent and unregulated angiogenesis is characteristic of tumor growth and it supports the pathological damage seen in cancer. Thus, tumor growth is an angiogenesis-dependent process (Folkman, 1971). After an initial prevascular phase, every increase in tumor endothelial cell population is preceded by an increase in new capillaries converging on the tumor. Expansion of tumor volume beyond this phase requires the induction of new capillary blood vessels.
Blockade of the angiogenic process has emerged as an important anticancer strategy. It has been demonstrated that in mice bearing subcutaneous Lewis lung carcinomas (3LL), the primary or local tumor releases an angiogenesis-inhibiting substance, named angiostatin (O""Reilly, et al., 1994). Angiostatin is a 38-kDa fragment of plasminogen that selectively inhibits proliferation of endothelial cells. Angiostatin has been shown to suppress vascularization and, hence, growth of metastases when used as an adjuvant to conventional therapy (e.g. see, U.S. Pat. No. 5,733,876). Several studies have produced results consistent with this model. After systemic administration, purified angiostatin can produce apoptosis in metastases (Holmgren, et al., 1995) and sustain dormancy of several human tumors implanted subcutaneously in nude mice (O""Reilly et al., 1996).
Clearly, angiogenesis plays a major role in tumor development and maintenance. As stated earlier, conventional cancer therapeutic regimens are hampered by the ability of the cancer cell to adapt and become resistant to the therapeutic modality used to combat tumor growth. Certain isocoumarin derivatives are known to have an inhibiting effect on vascularization (e.g. see, U.S. Pat. No. 6,020,363 which is incorporated herein by reference), and it is likely that such isocoumarin derivatives will be useful in reducing the growth, size, spreading, and otherwise mitigating the deleterious effect of a tumor by virtue of their angiogenesis-inhibiting properties. Significantly, pursuant to the present invention, isocoumarin derivative treatment has also been shown to sensitize endothelial cells and established tumors to the cytotoxic effects of ionizing radiation and/or conventional chemotherapy. Thus, the combination of these properties of isocoumarin derivatives provides for a promising cancer therapeutic strategy and establishes a novel medical application for these drugs.
An isocoumarin derivative, particularly 3-hydroxymethyl-6-methoxy-8-hydroxy-1H-2-benzopyran-1-one, subsequently named cytogenin (formula I, below), was first identified as a compound produced by the M143-37F11 strain of Streptoverticillium eurocidicum. Cytogenin has attracted attention under the name of the antibiotic M143-37F11 because of its growth-inhibiting activity against various animal cells and human cancer cells (Japanese Patent Laid-Open No. 2177/91). Cytogenin given by oral administration demonstrated antitumor activities against several transplantable mouse solid tumors. However, this compound showed very low anti-proliferation inhibitory activity against cultured tumor cells and low toxicity in mice and dogs. The mechanism of action of antitumor activity of cytogenin was found to be associated with its ability to disrupt the neovascularization in growing tumors. While cytogenin demonstrated promise as an antitumor agent, its in vivo instability rendered it unfit for clinical development. Investigations of chemical derivatives of cytogenin identified a highly stable analog, herein referred to as NM-3 (formula II, below) that retained the important biological properties of the parent compound. NM-3 is the most extensively studied member of this class of compounds (e.g. see, U.S. Pat. No. 6,020,363), and will serve as the primary example of an isocoumarin derivative useful in the treatment methods of the present invention. 
In its broadest aspects, the present invention is directed to the use of isocoumarin derivatives of formula III, below, in combination with ionizing radiation and/or chemotherapeutic agents in the treatment of cancer to provide a therapeutic effect, in terms of reduced cancer mass or metastatic potential, which is enhanced over that obtained with radiation and/or the chemotherapeutic agent alone.
The isocoumarin derivatives useful in the methods of the invention include compounds of formula III: 
wherein each of X1, X2, and X3 may independently be sulphur or oxygen; and X4 is sulfur, oxygen or xe2x80x94NHxe2x80x94; R1 is hydrogen or C1-6 alkyl, and R2 and R3 may be the same or different and each represents C1-6 alkyl, C7-10 alkaryl, hydroxy or hydrogen, and n is zero or a whole integer of from 1 to 3. The isocoumarin derivatives of the formula III function as radiation sensitizers and/or chemopotentiators, providing therapeutic benefit in the treatment of cancer which is additive or synergistic over that obtained with the isocoumarin derivative on the one hand, or ionizing radiation on the chemotherapeutic agent on the other hand, when either is used as the single treatment modality.
Accordingly, the present invention provides a method of inhibiting the growth of a tumor cell comprising contacting said tumor cell with an isocoumarin derivative of formula III, above, and a second growth inhibiting modality (or mode of treatment) selected from a dose of ionizing radiation or a chemotherapeutic agent or combination of said radiation and chemotherapeutic agent, wherein the does of said isocoumarin derivative, when combined with the second growth inhibiting modality, is effective to inhibit growth of said tumor cell.
In one embodiment, the invention further provides a method of treating cancer in a human patient comprising administering an isocoumarin derivative of formula III, above, to a human patient together with a dose of ionizing radiation and/or a chemotherapeutic agent, wherein the dose of said isocoumarin derivative, when combined with the dose of ionizing radiation and/or chemotherapeutic agent, is effective to treat the cancer. In this embodiment the cancer subject to treatment is suitably selected from the group consisting of skin cancer, prostate cancer, lung cancer, brain cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, pancreatic cancer, colon cancer, stomach cancer, lymphoma and leukemia. In the method of the invention, the isocoumarin derivatives are suitably administered prior to, or simultaneously with, the ionizing radiation and/or chemotherapeutic agent. The invention further provides a method of potentiating the effect of ionizing radiation on a tumor cell comprising contacting the tumor cell with an isocoumarin derivative of formula III, above, and then contacting the tumor cell with ionizing radiation, as well as a method of sensitizing a tumor cell to the growth inhibiting or cell killing effects of a chemotherapeutic agent, comprising contacting the tumor cell with an isocoumarin derivative of formula III, above, and then contacting the tumor cell with the chemotherapeutic agent.
Further, as used herein, chemotherapeutic agent includes all conventional cytotoxic and cytostatic agents used in cancer treatment and prevention including, from a mechanism of action standpoint; Tubulin interactive agents, DNA-interactive agents, antimetabolites and antifolates, antihormonals, antibiotics, antivirals, ODC inhibitors, and other cytotoxic agents, and prodrugs.